title: 21-Methylpyrenyl-cholesterol stably and specifically associates with lipoprotein peripheral hemi-membrane: a new labelling tool.
abstract: Lipoproteins are important biological components. However, they have few convenient fluorescent labelling probes currently reported, and their physiological reliability can be questioned. We compared the association of two fluorescent cholesterol derivatives, 22-nitrobenzoxadiazole-cholesterol (NBD-Chol) and 21-methylpyrenyl-cholesterol (Pyr-met-Chol), to serum lipoproteins and to purified HDL and LDL. Both lipoproteins could be stably labelled by Pyr-met-Chol, but virtually not by NBD-Chol. At variance with NBD-Chol, LCAT did not esterify Pyr-met-Chol. The labelling characteristics of lipoproteins by Pyr-met-Chol were well distinguishable between HDL and LDL, regarding dializability, associated probe amount and labelling kinetics. We took benefit of the pyrene labelling to approach the structural organization of LDL peripheral hemi-membrane, since Pyr-met-Chol-labelled LDL, but not HDL, presented a fluorescence emission of pyrene excimers, indicating that the probe was present in an ordered lipid micro-environment. Since the peripheral membrane of LDL contains more sphingomyelin (SM) than HDL, this excimer formation was consistent with the existence of cholesterol- and SM-enriched lipid microdomains in LDL, as already suggested in model membranes of similar composition and reminiscent to the well-described "lipid rafts" in bilayer membranes. Finally, we showed that Pyr-met-Chol could stain cultured PC-3 cells via lipoprotein-mediated delivery, with a staining pattern well different to that observed with NBD-Chol non-specifically delivered to the cells.
authors.affiliation: INSERM U563, CHU Purpan, Toulouse, France; CEA, SB2SM and UMR8221 CNRS, IBiTec-Saclay, Gif-sur-Yvette, France.
authors.forename: Gérald
authors.initials: G
authors.lastname: Gaibelet
authors.forename: François
authors.initials: F
authors.lastname: Tercé
authors.forename: Justine
authors.initials: J
authors.lastname: Bertrand-Michel
authors.forename: Sophie
authors.initials: S
authors.lastname: Allart
authors.forename: Vincent
authors.initials: V
authors.lastname: Azalbert
authors.forename: Marie-France
authors.initials: MF
authors.lastname: Lecompte
authors.forename: Xavier
authors.initials: X
authors.lastname: Collet
authors.forename: Stéphane
authors.initials: S
authors.lastname: Orlowski
mesh_terms: D045744:Cell Line, Tumor; D002478:Cells, Cultured; D002784:Cholesterol / Q000031:analogs & derivatives* / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D008075:Lipoproteins, HDL / Q000097:blood / Q000737:chemistry; D008077:Lipoproteins, LDL / Q000097:blood / Q000737:chemistry*; D008566:Membranes / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013194:Staining and Labeling
chemical_list: D005456:Fluorescent Dyes; D008075:Lipoproteins, HDL; D008077:Lipoproteins, LDL; D002784:Cholesterol
keywords: (V)LDL; (very) low-density lipoproteins; Chol; Cholesterol; Fluorescence; HDL; LCAT; Lipid monolayer; Lipoproteins; Membrane microdomains; NBD; Pyr-met; SM; Sphingomyelin; apo; apolipoprotein; cholesterol; high-density lipoproteins; lecithin:cholesterol acyltransferase; methylpyrenyl; nitrobenzoxadiazole; sphingomyelin